Poly (ADP-ribose) polymerase is a chromatin-bound, protein-modifying enzyme found in the nuclei of all eukaryotic cells. Using NAD as a substrate, the enzyme ADP-ribosylates a variety of chromosomal proteins, including histones, a Mg++-, Ca++-dependent endonuclease, and (in HeLa cells) a 5.2S, 112K nonhistone chromosomal protein tentatively identified as poly (ADP-ribose) polymerase. Considerable circumstantial evidence suggests that poly (ADP-ribose) polymerase activity is required for one or more of the processes of DNA repair in eukaryotic cells, though perhaps through a more fundamental activity concerned with the regulation of chromatin structure. The experimental protocol outlined in this proposal is intended to better elucidate the role of this enzyme in the cellular recovery from damage to DNA elicited by both UV-light and the monofunctional alkylating agent, N-methyl-N-nitrosourea (MNU). Specifically, the activity of poly (ADP-ribose) polymerase, as well as the profile of its modified acceptors, will be examined in asynchronous and synchronous HeLa cells, both under normal (control) conditions, and following treatment of cells with the above mutagens. In the synchronous cell culture studies, enzyme activity and acceptor profiles will be examined in the G1, mid-S, S-G2, G2 and M stages of the cell cycle. In addition, poly (ADP-ribose) polymerase activity will be assayed in xeroderma pigmentosum (XP) fibroblasts from complementation groups A, C and D, which have been reported capable of excising pyrimidine dimers from naked DNA, but not chromatin, and which reportedly have lowered levels of NAD utilizaton following mutagenesis. Attempts will be made to restore the repair capability of extracts of these mutunt cells through the exogenous addition of poly (ADP-ribose) polymerase purified from normal fibroblasts and HeLa cells. ADP-ribosylated acceptor protein profiles in normal and XP fibroblasts will also be examined to determine if a single and critical acceptor is not being modified, perhaps because of a mutant site of ADP-ribosylation. Finally, a variety of inhibitors of poly (ADP-ribose) polymerase, including NADP, nicotinamide, thymidine, caffeine and benzamide, will be assayed for their effects on DNA repair.